The transformation of neoplastic disease to a highly tumorigenic, metastatic, and radio / chemotherapy resistant phenotype is a result of the accumulation of a number of genetic changes. Combination therapies have emerged as powerful alternatives to `single-agent therapies'since they enhance cell death by overcoming multiple resistance mechanisms in cancer cells. Tumor Necrosis Factor-1 Related Apoptosis Inducing Ligand (TRAIL) and agonistic antibodies to Death Receptor (DR) 4 and 5 have attracted significant attention due to their ability to selectively induce apoptosis in transformed (malignant) cells while demonstrating little cytotoxicity in normal cells. Despite this promise, many tumor cells are resistant to TRAIL and as a result, radiation and chemotherapeutic genotoxins have been employed to sensitize cancer cells to death receptor- mediated apoptosis. While this approach has been moderately successful, traditional `single-chemosensitizer discovery'approaches are inherently low-throughput and do not allow for comparison of different sensitizing agents. We hypothesize that screening small molecule libraries can result in the rapid identification of new combination treatment strategies and candidates and enable comparison of different molecules. In this proposal, we describe a novel cancer cell-based screening paradigm in which we integrate traditional well plate based screening and microfabrication / microfluidics technologies for the identification and mechanistic evaluation of small-molecules that sensitize cancer cells to death receptor-mediated apoptosis. Well-plate screening methods will be employed in the primary screening of a library of ~2,500 FDA approved drugs for identifying lead candidates. Microfluidic cancer cell arrays will then be employed to carry out stringent secondary screening of leads selected from the primary screen;it is hypothesized that the ability to interrogate cells with combinatorial stimuli using microfluidics can lead to the parallelization and miniaturization of the secondary screening step. The higher throughput required in the primary screening and the combinatorial dosing requirements in the secondary screening justify our choices of using conventional 96-well plates for the former and a microfluidcs-based cancer cell array for the latter. It is anticipated that the proposed research will lead to the development of a novel integrated screening paradigm based on well-plates and microfluidic cell arrays for the discovery of small molecules for combination treatments and the identification and mechanistic evaluation of approximately 20-50novel candidates that enhance cancer cell death in combination with death receptor agonists. The proposed screening platform has implications beyond the present work;the platform can be extended to screen/investigate various factors influencing combination therapies including, other therapeutic candidates (e.g. peptides, siRNA, etc.), soluble factors (e.g. cytokines, metabolites, etc.), and tumor microenvironment effects. Project Narrative This proposal aims to identify effective combination treatments for cancer therapy using high-throughput screening. A novel HTS paradigm based on the integration of traditional well-plate screening and a microfluidic cancer cell array will be used. The microfluidic cell array will be developed as a platform for investigating synergistic interactions between candidate molecules. Successful completion of this work can lead to the development of new combination anti-cancer therapies and in other cell-based high- throughput screening applications. PUBLIC HEALTH RELEVANCE: This proposal aims to identify effective combination treatments for cancer therapy using high-throughput screening. A novel HTS paradigm based on the integration of traditional well-plate screening and a microfluidic cancer cell array will be used. The microfluidic cell array will be developed as a platform for investigating synergistic interactions between candidate molecules. Successful completion of this work can lead to the development of new combination anti-cancer therapies and in other cell-based high through put screening applications.